A collaborative, multidisciplinary approach will be applied to the study of the first step in poliovirus genome replication, negative-strand RNA synthesis. Given the wealth of information already available on the molecular biology of poliovirus, it is easy to assume that everything is known about poliovirus genome replication. Indeed, vivid, colorful, detailed models of this process appear in every virology textbook. However, the recent discovery that initiation of poliovirus negative-strand RNA synthesis occurs in the center of the genome rather than at the 3' end calls into question previous models. Moreover, our studies of poliovirus genome replication have uncovered the existence of a novel interaction between key replication proteins that is essential for initiation of negative-strand RNA synthesis and challenge the dogma that the processed forms of the replication proteins which accumulate within infected cells are, indeed, the same forms of the proteins required for establishment of the initiation complex. The central hypothesis driving these studies is as follows. Unprocessed forms of the replication proteins bind first to the cis-acting replication elements located at the extreme 5' and 3' ends of the genome, folding into a ribonucleoprotein complex that may function to turn off translation of the genome. Binding of precursor proteins to the internal site of initiation recruits the ends of the genome to the middle. The protein primer is then uridylylated by polymerase, and the two-nucleotides-extended primer is then efficiently transferred to the 3' end owing to its preexistence in this complex. Continued synthesis results in the production of full-length, negative-strand RNA. We will begin testing this hypothesis by pursuing the following specific aims: (1) Evaluate the interaction of unprocessed forms of PV replication proteins with the cis-acting replication elements (CREs): oriL, oril and oriR; (2) Define the molecular basis for the specificity of the interaction between P3 proteins and the various CREs; (3) Define the surface of protein 3C used to recruit polymerase to the initiation complex by using a combination of reverse-genetic and classical genetic approaches; (4) Develop a system to study initiation of negative-strand RNA synthesis (i.e., VPg priming) in vitro that functions on a biologically relevant time scale. Poliovirus remains one of the most intensively and extensively studied members of the Picornavirus family. This family includes viruses that cause myriad human diseases, including the common cold, hepatitis and chronic, inflammatory heart disease. These studies will provide a more precise understanding of the molecular mechanism of picornavirus genome replication. Protein-RNA and protein-protein interactions identified in this study that are critical for genome replication represent targets for chemotherapeutic intervention. Therefore, this information may prove useful in the development of strategies to control picornavirus infections for which vaccines are not available.